Antibacterial and Anticandidal Activity of the Nanostructural Composite of a Spirothiazolidine-Derivative Assembled on Silver Nanoparticles

Our aims in this work are the preparation of an ionic liquid based on heterocyclic compounds with Ag nanoparticles and the investigation of its application as an antibacterial and anticandidal agent. These goals were achieved through the fabrication of an ionic liquid based on Ag nanoparticles with 5-Amino-3-(4-fluorophenyl)-N-hexadecyl-7-(4-methylphenyl)-2-H spiro[cyclohexane1,2’-[1,3]thiazolo [4,5-b]pyridine]-6-carbonitrile (P16). The nanostructure of the prepared ionic liquid was characterized using techniques such as FTIR, 1HNMR, 13CNMR, UV, SEM, and TEM. The biological activity of the prepared compound (P16) and its nanocomposites with Ag nanoparticles was tested using five clinical bacteria (Pseudomonas aeruginosa 249; Escherichia coli 141; Enterobacter cloacae 235; Staphylococcus epidermidis BC 161, and methicillin-resistant S. aureus 217), and three Candida species (Candida utilis ATCC 9255; C. tropicalis ATCC 1362, and C. albicans ATCC 20402). The FTIR, 1HNMR, and 13CNMR results confirmed the chemical structure of the synthesized P16 compound. The nanostructure of the prepared ionic liquid was determined based on data obtained from the UV, SEM, and TEM tests. The antibacterial and anticandidal results showed that the biological activity of the compound (P16) was enhanced after the formation of nanocomposite structures with Ag nanoparticles. Moreover, the biological activity of the compound itself (P16) and that of its nanocomposite structure with Ag nanoparticles was higher than that of ampicillin and amphotericin B, which were used as control drugs in this work.


Introduction
Ionic liquids are among the most important charged surface activity agents.Recently, scientists have become interested in developing environmentally friendly, safe, and effective synthesis methods to achieve the best results in this field.Ionic liquids have been reported as green solvents and alternatives to VOCs.The characteristics of ionic liquids (ILs) are high stability, high conductivity, low vapor pressure, high temperature, and their ability to dissolve polar and nonpolar compounds [1].They are used in organic synthesis, catalysis, food science, analytical chemistry, and nanotechnology.Also, ionic liquids are important in medicinal chemistry; due to their biological properties they are used as disinfectants, antimicrobials, and anti-inflammatory and anti-cancer agents [2].The development, amalgamation, and production of molecules with benefits for people are among the basic objectives of therapeutic natural chemistry [3][4][5].Recent developments in combinatorial chemistry have made it possible to obtain chemical libraries based on desirable structures [6][7][8].A class of naturally occurring chemicals known as spiro-compounds has important biological characteristics [9][10][11]; due to the link between their structure and activity, they are a particularly engaging target for the synthesis of combinatorial libraries [12][13][14].This group represents a sizeable class of heterocycles with nitrogen and sulfur that are commonly utilized as important building units within the pharmaceutical field [15][16][17].Recently, an effective and additive-free green method was developed for the environmentally friendly synthesis of heterocycles with amazing yields and quick reaction times [18][19][20][21][22].A literature survey revealed that thiazolidine is a parent compound of an array of substances that are of fundamental importance in restorative and pharmaceutical chemistry.Key medications, including pioglitazone, epalrestat, letosteine, and tidiacic, are among those that integrate the thiazolidine nucleus, which is significant in medical and pharmaceutical chemistry (Scheme 1).The nucleus of thiazolidine is also referred to as the "wonder nucleus", since it produces a variety of derivatives with a wide range of biological functions [23][24][25].It has been demonstrated that this compound possesses antibacterial and anti-HIV [26,27] properties due to the presence of a N-C-S linkage.Spirothiazolidines have received less attention, even though they enable conformational qualifications and original orientation of a variety of elements in spiro-compounds.This platform is recognized as a valuable isostere for known dynamic spiroimidazolidines, spirohydantoines, and spirobenzofuranes, with usage in G protein-coupled receptors (GPCRs) and peptidomimetics.It is a component of the antipsychotic medication spiclomazine.Simple spirothiazolidines have been shown to inhibit metalloprotease, giving rise to their use in the management of malaria infections [28].Derivatives of spirothiazolidine have also been shown to have good antioxidant [29], anticancer [30], antihyperglycemic, antidiabetic [31], anti-inflammatory [32,33], anticonvulsant [34][35][36], and antimicrobial activities [37].In this respect, nanomaterials have been receiving increased attention in recent years.Notably, silver nanomaterials show a noteworthy contrast to other macroscopic and small-molecular species due to their particle sizes.They have broad applications within the chemical industry, i.e., in medications, biological agents, superconductors, and other fields [38].Considering these advantages, the primary goals of our work are to synthesize a biologically important scaffold based on spiro(cyclohexane-thiazolidine) derivatives and related nanocomposites and to evaluate the antibacterial and antifungal activity of these materials.

Nanostructure Formation of the P16 with AgNPs (P16-AgNPs)
The first technique used in this work to establish the nanostructure of P16 with Ag-nanoparticles was based on the UV spectrum.The UV spectrum of the prepared Agnanoparticles is shown in Figure 2.This figure shows the excitation and emission spectra of the Ag-nanoparticles at ~422 nm −1 .This peak appeared because of the surface plasmon absorption of silver clusters [39].The UV results of the nanostructure of the synthesized P16 with the Ag-nanoparticles are represented in Figure 2.These results reveal that the peak of the AgNPs at 422 nm completely disappeared; this was due to the reduction of the negative charge that surrounded the particles as result as of the assembly of P16 molecules on AgNPs; it is known that metal particles in aqueous colloidal dispersions usually have a negative charge due to the adsorption of anions.The addition of neutral molecules such as P16 neutralizes the adsorbed ions and reduces the charge on these particles.In addition, the presence of P16 on AgNPs formed nano shells instead of the nanoparticles and prevented the aggregation of AgNPs [39].
The second technique used in this work to establish the nanostructure of the P16 with Ag-nanoparticles was TEM. Figure 3 presents TEM micrographs of the individual AgNP solution and AgNPs in the presence of P16.The nanostructure of the AgNPs in Figure 3 can be seen to comprise a spherical shape and polycrystalline structure, with an average size ranging from 19.1 to 21.44 nm.After capping the P16 molecules on the AgNPs, nanoshells were formed, restricting the aggregation of AgNPs due to the formation of nanoshells with P16 molecules (see Figure 3).In addition, the assembly of P16 molecules increased the stability of the AgNPs and reduced their aggregation.Therefore, the presence of alkyl chains (hexadecyl) in compound P16 enhanced the stability of the AgNPs.It was concluded that the alkyl chain length provided a physical barrier, preventing aggregation between the surfaces of AgNPs during collisions, in addition to the effect of P16 molecules on the charge of AgNPs; that is, the P16 reduced the surface charge of the AgNPs [40].These factors led to an overall reduction in the aggregation of AgNPs, as shown in Figure 3.The SEM images in Figure 3 were used to further investigate the formation of the nanostructure of AgNPs and P16-AgNPs.It is clear from the SEM image presented in Figure 3 that the sizes of the AgNPs ranged from 43.7-51.7 nm.Moreover, the SEM image in Figure 3 gives an indication about the aggregation of the Ag-nanoparticles which were produced as a result of the surface plasmon charges on the silver nanoparticles.It is known that metal particles in colloidal solution usually have a negative charge due to adsorbed anions; this leads to more aggregation of such particles in solution, as shown in Figure 3 [40].Further investigation of the nanostructure of the P16 capped AgNPs (P16-AgNPs) is summarized in Figure 3.It was noted from previous publications that the aggregation of the nanoparticles can be weakened by mixing with neutralized molecules such as P16, as these neutralize the adsorbed ions and reduce the charge on the particles, thereby preventing the particles from aggregating with each other [40].The SEM images in Figure 3 reveal the formation of nanoshells of P16 after capping with AgNPs.This capping produced nanoshells instead of nanoparticles and prevented the aggregation of the Ag-nanoparticles, as shown in Figure 3.The second technique used in this work to establish the nanostructure of the P16 with Ag-nanoparticles was TEM. Figure 3 presents TEM micrographs of the individual AgNP solution and AgNPs in the presence of P16.The nanostructure of the AgNPs in Figure 3 can be seen to comprise a spherical shape and polycrystalline structure, with an average size ranging from 19.1 to 21.44 nm.After capping the P16 molecules on the AgNPs, nanoshells were formed, restricting the aggregation of AgNPs due to the formation of nanoshells with P16 molecules (see Figure 3).In addition, the assembly of P16 molecules increased the stability of the AgNPs and reduced their aggregation.Therefore, the pres- AgNPs (P16-AgNPs) is summarized in Figure 3.It was noted from previous publications that the aggregation of the nanoparticles can be weakened by mixing with neutralized molecules such as P16, as these neutralize the adsorbed ions and reduce the charge on the particles, thereby preventing the particles from aggregating with each other [40].The SEM images in Figure 3 reveal the formation of nanoshells of P16 after capping with AgNPs.This capping produced nanoshells instead of nanoparticles and prevented the aggregation of the Ag-nanoparticles, as shown in Figure 3.   1 and 2 and Figure 4.For cationic surfactants such as P16, it was found that the antimicrobial activity depended mainly on the aliphatic chain length; this phenomenon is known as the cut-off effect [41,42].Many parameters are dependent upon the occurrence of the cut-off effect, including the critical micelle concentration of the surfactant, a change in the free energy of the adsorption of the cationic surfactant on the bacterial cell membrane, the hydrophobic properties, and the size of the diffused surfactant [43].Optimal antimicrobial activity is achieved in medium chain length (C8-C18) cationic surfactants, while surfactants with alkyl chain less than C4 or more than C18 are mostly inactive [44].Moreover, in the medium chain length range, an increase in the aliphatic chain length of the surfactant decreases the critical micelle concentration, thus lowering the surfactant concentration at the cell membrane.Accordingly, the surfactant activity will be greater with an alkyl chain length ranging from 10 to 18 carbon atoms.On the other hand, the adsorption capacity at the membrane interface should increase with increasing the hydrophobicity of the surfactant.Gram-negative bacteria are more antibiotic-resistant than Gram-(+ev) bacteria, yeast, and fungi.The outer membrane, which is composed of lipopolysaccharide molecules and periplasmic space in the Gram (-ev) bacterial cell, prevents the penetration of antibacterial agents, which make it more antibiotic-resistant [45][46][47].Our results, as shown in Tables 1 and 2 and Figure 4, are in clear agreement with the previously mentioned phenomena (cut-off effect) [41,42], as the activity of P16-AgNPs against the five clinical bacteria and the three Candida species under investigation was higher than that of P16 without AgNPs.In addition, the biological activity of P16 alone and its nanostructures with Ag-nanoparticles was higher than that of the Ampicillin or amphotericin B, which were used as control drugs.Generally, the results were good; after the formation of a nanostructure of P16 with AgNPs, its biological activity clearly improved.As detailed in Tables 1 and 2, the inhibition zone of P16 increased after capping with the AgNPs.It can be seen from the results in Tables 1 and 2 and Figure 4 that the biological activity of P16 toward most bacteria and candida strains was high.This may be related to the fused pyridine ring with spirothiazolidine in the chemical structure of P16, which this likely played an important role in enhancing the biological activity [37].Moreover, nucleophilic groups (NH 2 , CN) and the presence of electron-donating groups (CH 3 , hexadecyl) likely also resulted in the high degree of biological activity of P16 [37].These results indicate that P16 favored the dispersion of AgNPs and improved their biocompatibility.The inhibitory effect of the capped AgNPs may be attributed to the generation of free radicals and Reactive Oxygen Species (ROS) in microbial cells, leading to the disruption of the cell membrane and damage to cellular proteins, as shown in Figure 5 [48].The inhibition zone around the discs impregnated with P16 and P16AgNPs is expressed as the mean of three replicates (mm ± SD).SD: standard deviation.a-d, A-C: Each value represents an average of three repetitions.The means followed by the same letters were not significantly different at p = 0.05, based on Duncan's multiple-range test.Small letters are used to compare the mGIZ of the compound, compound-AgNPs, and the control drugs with different strains, while capital letters are used to compare the mGIZ of the compound P16, P16-AgNPs, and the control drugs for the same strain.3.1.1.Synthesis of 4-(4-Fluorophenyl)-1-thia-4-azaspiro [4,5]decan-3-one (Compound 1) Compound 1 was prepared by the reaction of cyclohexanone (0.01 mol), p-fluoroaniline (0.01 mol), and thioglycolic acid (0.01 mol) in dry toluene (50 mL).This mixture was then refluxed for 10 h.After filtration, drying, and recrystallization of the product from the dioxane/methanol mixture, pale-yellow needles of compound 1 were obtained [29].For the preparation of compound 2, compound 1 (0.01 mol), p-tolualdehyde (0.01 mol), ammonium acetate (0.02 mol), and malononitrile (0.01 mol) in glacial acetic acid (40 mL) were refluxed for 24 h.This mixture was cooled and poured into water to obtain a solid precipitate.After filtration, drying, and recrystallization from dioxan, a deep yellow powder comprising compound 2 formed [29].
A solution of compound 2 (0.02 mol) in 50 mL DMF was prepared at room temperature.To this was added 1-bromhexadecane (0.02 mol).The mixture was then heated to 100 • C for 6 h.After cooling and the addition of diethyl ether, the product was precipitated.The crude precipitate was recrystallized twice from methanol to yield 5-Amino-3-( 4

Synthesis of Silver Nanoparticles (AgNPs)
An Ag-nanoparticle colloidal solution was synthesized using the chemical reduction method shown in previous publication [40].Briefly, 0.01 g AgNO 3 was placed in 50 mL of distilled water the solution was boiled to 100 • C.Then, 5 mL of 0.01 g 1% sodium citrate solution was added with stirring and the solution was heated to mix the components until the color changed to pale yellow.Then, heating was stopped and the solution was continuously stirred and cooled to room temperature.

Nanostructure of AgNPs with Compound P16 (P16-AgNPs)
A nanostructure of 5-Amino-3-(4-fluorophenyl)-N-hexadecyl-7-(4-methylphenyl)-2-Hspiro[cyclohexane-1,2'- [1,3] thiazolo [4,5-b]pyridine]-6-carbonitrile (P16) with Ag-nanoparticles was fabricated according to the following method: a solution of P16 was prepared by dissolving it in propanol solution under continuous stirring for 1 h.Then, 20 mL of Agnanoparticles aqueous solution was mixed with 5 mL of the P16 solution.After that, the mixture was stirred until the pale yellow AgNPs became colorless [40].The FTIR spectra of the prepared P16 was determined at the College of Science, University of Ha'il, Ha'il, KSA using a Thermo Nicolet 6700 FT-IR optical spectrometer (Mundelein, IL 60060, USA).The proton nuclear magnetic resonance ( 1 HNMR) spectra of the synthesized compound (P16) were determined using a 850 MHz Ascend 850 Mhz NMR spectrometer (Bruker, Billerica, MA, USA).Tetramethylsilane was used as an internal reference and DMSO as a solvent.The samples were measured at the College of Science, University of King Abdulaziz, Jeddah, KSA.In addition, the chemical structure of the synthesized P16 was confirmed using 13 CNMR spectra data.Analyses were carried out using an 850 MHz Ascend 850 Mhz NMR spectrometer (Bruker, Billerica, MA USA).Tetramethylsilane was used as an internal reference and DMSO as a solvent.Analyses were carried out at the College of Science, University of King Abdulaziz, Jeddah, KSA.

Conclusions
Ionic liquids (ILs) have attracted interest due to their antibacterial and antimicrobial activities.Recently, nanomaterials have also received considerable attention from researchers.The most important of these nanomaterials are based on silver.In the present work, we investigated the fabrication of ionic liquids based on Ag-nanoparticles with 5-Amino-3-(4-fluorophenyl)-N-hexadecyl-7-(4-methylphenyl)-2-H-spiro[cyclohexane-1,2'- [1,3]thiazolo [4,5-b]pyridine]-6-carbonitrile (P16).In addition, we studied the biological activity of the fabricated ionic liquids against five clinical bacteria and three Candida species.The overall results confirmed that the fabricated ionic liquid had high as antibacterial and antimicrobial activities.The P16AgNPs showed higher activity against the five clinical bacteria and the three Candida species than P16 alone.Moreover, the efficiency of P16AgNPs was higher than that of the Ampicillin and amphotericin B, which were used as control drugs.

Molecules 2024 , 15 Scheme 1 .
Scheme 1. Examples of common drugs based on the thiazolidine ring.

Scheme 1 .
Scheme 1. Examples of common drugs based on the thiazolidine ring.

Figure 4 .P . a e r u g i n o s a ( 2
Figure 4. Comparison of the effect of P16, P16-AgNPs, and the control drugs (ampicillin and amphotericin B) on the growth inhibition of five clinical bacteria (Pseudomonas aeruginosa 249; Escherichia coli 141; Enterobacter cloacae 235; Staphylococcus epidermidis BC 161, and Methicillin-Resistant S. aureus 217) and three Candida species (Candida utilis ATCC 9255; C. tropicalis ATCC 1362, and C. albicans ATCC 20402).

Figure 5 .MFigure 5 .Scheme 2 .Scheme 2 .
Figure 5. Disruption of the cell membrane and damage to cellular proteins with P16 and P16AgNPs.

Table 1 .
Antibacterial and anti-Candida activities of P16 and P16-AgNPs, as compared to those of ampicillin and amphotericin B, tested using disc diffusion assay.

Table 2 .
Determination of MICs and MBCs/MFCs values using microdilution assay for both P16 and P16-AgNPs.